Oligosaccharide inhibition of Streptococcus pyogenes adhesion

ABSTRACT

A method of, and oral composition for, inhibiting adherence of Streptococcus pyogenes to pharyngeal and oral mucosa cells by treating these cells with an oligosaccharide having at least one fucose moiety or a galactose moiety which is free of digalactose and N-acetylneuraminyl lactose.

FIELD OF THE INVENTION

This invention relates to a method of inhibiting adherence and promotingdesorption of Streptococcus pyogenes to pharyngeal and oral mucosa cellsby treating these cells with an oligosaccharide.

SUMMARY OF THE INVENTION

The object of this invention is to inhibit the attachment ofStreptococcus pyogenes to human epithelial cells by treating pharyngealand oral mucosal cells with an oligosaccharide.

Another object of the invention is to incorporate an adhesion inhibitingoligosaccharide into a preparation for oral application.

Conventional therapy for S. pyogenes infections has mainly consisted oftreatment with antibiotics such as penicillin and tetracycline.Unfortunately, antibiotics such as penicillin and tetracycline exhibitbroad spectrum antimicrobial activity. Thus, treatment with theseantibiotics tends to kill not only S. pyogenes but a number of otherbacterial species, some of which may actually be beneficial to the body.In contrast, the present invention, by using oligosaccharides tospecifically inhibit the adherence of S. pyogenes to pharyngeal and oralmucosal cells, does not disturb the normal microbial ecology of themouth. Further, many oligosaccharides are derived from natural sourceswhich are endogenous, and therefore unlikely to be, distruptive to thebody.

BACKGROUND OF THE INVENTION

Adherence is an important early event in the pathogenesis of bacterialinfections in animals and humans. Studies have shown that the infectiousability of bacteria is related to the ability of bacteria to adhere tohost cells. In the first stages of infection, bacterial adhesins,adhesive molecules on the surface of bacteria, bind to receptormaterials on the host cell membrane.

Surprisingly, it has been found that the adherence of a bacterium suchas Streptococcus pyogenes to human epithelial pharangeal and oralmucosal cells can be inhibited by oligosaccharides. StreptococcusPyogenes, a group A beta hemolytic streptococci which causespharyngitis, exhibits tissue tropism, i.e. it is virtually found only inhumans.

Early studies by Ellen and Gibbons (Infection and Immunity, pp. 826-830May 1972) indicated that M protein functions in the attachment of S.pyogenes to epithelial surfaces. On the other hand, subsequent studiesby Beachey (J. of Infectious Diseases 143(3), pp. 325-345(1981))indicated that attachment of S pyogenes to host receptor cells occurredthrough fatty acid ends of lipoteichoic acid molecules on the S.pyogenes surface interacting with host cell membrane receptors. Morerecently Tylewska et al (Current Microbiology,16, pp. 209-216(1988))have confirmed that the M protein in the S. pyogenes cell membrane mayplay an important role in S. pyogenes adhesion to host cell membranes.While the inventors do not wish to be bound to any one theory, it isbelieved that inhibition by oligosaccharide of the adherence of S.pyogenes to human pharyngeal and oral mucosal cells may be accomplishedby the oligosaccharide effectively mimicing the host cell receptors forM protein, thus preventing binding of S. pyogenes to the receptors of Mprotein on the host cell membrane.

DETAILED DESCRIPTION

The present invention relates to the inhibition of S. pyogenes adhesionto human pharyngeal and oral mucosal cells by oligosaccharides.

The oligosaccharide contains two or more, preferably 3 or more sugarresidues, at least one of which is a galactose moiety free ofdigalactose and N-acetylneuraminyl lactose, or a fucose moiety. Itpresently appears that the exact linkage between the galactose moiety orfucose moiety and other sugar residues does not affect the utility ofthe oligosaccharides of the invention.

Preferred oligosaccharides include:

    ______________________________________                                        B-trisaccharide                                                               Gal α 1-3 Gal                                                                      2                                                                             |                                                                  Fuc α 1,                                                       A-Tetrasaccharide                                                             Gal-NAc 1-3 Gal β 1-4 GLc                                                           2                                                                             |                                                                  Fuc α 1,                                                       2 fucosyllactose, Fuc α 1-2 Gal β 1-4 GLc                          2 fucosyllactose, Gal β 1-4 GLc                                                     3                                                                             |                                                                  Fuc α 1                                                        Lacto-N-Tetrose                                                               Gal β 1-3 GLc Nac 1-3 Gal β 1-4 GLC                                 and Lactodifucotetraose                                                       Fuc α 1-2 Gal β 1-4 GLc                                                       3                                                                             |                                                                  Fuc α 1                                                        ______________________________________                                    

The effect of oligosaccharides on the adhesion of S. pyogenes to humanepithelial pharyngeal and oral mucosal cells of S. pyogenes isdetermined according to the following method. Epithelial cells areobtained by gently scraping the pharangeal, buccal or tongue mucosalsurfaces of healthy adults. The cells are suspended in 0.01M phosphatebuffer (pH 7.2) containing 0.1% bovine serum albumin (BSA) and washedtwice by centrifugation. The suspension is adjusted to containapproximately 5×10⁵ cells/ml. The attachment of S. pyogenes to theepithelial cells is determined by admixing ³ H-labelled S. pyogenes withthe epithelial cells. The mixture is then centrifuged in the presence ofPercoll, a colloidal Polyvinylpyrrolidone coated silica (available fromSigma Chemical Corporation) to form a density gradient. Under theseconditions, epithelial cells with attached bacteria form a tight bandnear the top of the centrifuge tube while unattached streptococci form aband near the bottom. The number of bacteria associated with epithelialcells is determined by direct scintillation counting.

Various oligosaccharides were added to the S. pyogenes epithelial cellmixture. The measured decrease in relative absorption of S. pyogenes inthe presence of oligosaccharide is shown in the following table.

                  TABLE                                                           ______________________________________                                                         Concentration                                                                             Relative                                         Control          mg/ml       Adsorption %                                     ______________________________________                                        (.01M Phosphate Buffer/BSA                                                                     --          100                                              pH = 7.2                                                                      Lacto-N-tetraose 0.5         55                                               Lactodifucotetraose                                                                            0.5         54                                               B-Trisaccharide   0.25       60                                               A-Tetrasaccharide                                                                               0.25       62                                               3-fucosyllactose 0.5         56                                               ______________________________________                                    

These results show that the adherence of S. pyogenes to epithelialpharangeal, buccal, or tongue mucosal surfaces is significantlyinhibited by oligosaccharides.

Preparations containing oligosaccharides for oral application may be inany convenient form, such as a mouthrinse, tablet or lozenge, chewinggum, throat spray and the like. The oral application may also be in theform of elixirs, syrups or suspensions, for example, solutionscontaining 0.1 to 5% by weight oligosacchride, sugar and a mixture ofethanol, water, glycerol, propylene glycol and optionally aroma,saccharine and/or sodium carboxymethylcellulose as a dispersing agent.

A mouthrinse or mouthwash may be prepared by mixing a nontoxic alcoholand water vehicle with flavoring oil, nonionic surfactant, humectant,sweetener, color and optionally antibacterial antiplaque agent, forexample, cetyl pyridinium chloride, benzethonium chloride, andchlorhexidine.

The alcohol component of a mouthwash, typically present in an amount ofabout 5-25% by weight, is a nontoxic alcohol such as isopropanol orethanol, preferably utilizing denaturing components which also functionas flavoring agents. These flavoring agents are used in an amountbetween about 1% and 2% of the total alcohol content of the mouthwash.Water typically comprises at least about 50% by weight of a mouthrinseand humectant about 5-40% by weight. Generally, the ratio of water toalcohol is in the range of from about 1:1 to about 20:1 preferably from3:1 to 20:1 and most preferably from about 5:1 to 10:1 by weight. Thetotal amount of water-alcohol mixture in this type of preparation istypically in the range of from about 70 to about 99.9% by weight of thepreparation.

The mouthwash may also contain 0.5 to 5% of a water-soluble organicsurfactant, typically an amphoteric surfactant such as betaine, and mostpreferably a nonionic surfactant. Preferable nonionic surfactantsinclude condensation products of ethylene oxide with various reactivehydrogen-containing compounds reactive therewith having long hydrophobicchains (e.g. aliphatic chains of about 12 to 20 carbon atoms), whichcondensation products ("ethoxamers") contain hydrophilic polyoxyethylenemoieties, such as condensation products of poly (ethylene oxide) withfatty acids, fatty alcohols, fatty amides, polyhydric alcohols (e.g.sorbitan monostearate), and most preferably polypropyleneoxide (e.g.Pluronic materials).

Examples of mouthwash formulations which may be employed in the methodof the present invention are as follows:

    ______________________________________                                                        parts by weight                                               ______________________________________                                        ethyl alcohol   10.0                                                          glycerol        10.0                                                          flavor          0.4                                                           sodium saccharin                                                                              0.03                                                          nonionic surfactant                                                                           2.0                                                           oligosaccharide 0.1 to 5.0                                                    water           Q.S. to 100.sup. .sup.                                        ______________________________________                                    

The oligosaccharids of this invention can also be incorporated inlozenges or in chewing gum or other products, e.g. by stirring into awarm gum base or coating the outer surface of a gum base; illustrativeof which may be mentioned jelutone, rubber latex, vinylite resins, etc.,desirably with conventional filler materials such as plasticizers orsofteners, sugar or other sweeteners or carbohydrates such as glucose,sorbitol, sorbitol syrup, mannitol, xylitol, hydrogenated starchhydrolysate, and the like, and artificial sweeteners for example,aspartame and L-aspartic acid derived sweeteners, saccharin salts,acesulfame-K and the like, and the free acid form of saccharin, andprotein based sweeteners such as thaumatin.

A typical chewing gum may contain the following ingredients, in percentby weight based on the weight of the total gum formulation:

    ______________________________________                                        Ingredients       parts by weight                                             ______________________________________                                        Gum base          10-40                                                       Sucrose           50-75                                                       Corn syrup or Glucose                                                                           10-20                                                       Oligosaccharide   0.01 to 5                                                   Flavor            0.1-5                                                       ______________________________________                                    

An alternate chewing gum formulation is as follows:

    ______________________________________                                        Ingredients      parts by weight                                              ______________________________________                                        Gum base         10-50                                                        Binder           3-10                                                         Filler           5-80                                                         (Sorbitol, mannitol or                                                        combination thereof)                                                          oligosaccharide  0.01 to 5                                                    Flavor           0.1-5                                                        ______________________________________                                    

In certain sugarless gums, there is used as the binder ingredient asolution of sorbitol in water containing from about 10% to about 80%,preferably from about 50% to about 75% by weight of sorbitol in H₂ O. Inothers, there is used a gum acacia-in-water system containing from about30% to about 60%, preferably from about 45% to about 50% by weight ofgum acacia powder.

The ingredient referred to heretofore in the formulations simply as "gumbase" is susceptible to many variations. In general, a gum base isprepared by heating and blending various ingredients, such as naturalgums, synthetic resins, waxes, plasticizers, etc. in a manner well knownin the art. Typical examples of the ingredients found in a chewing gumbase are masticatory substances of vegetable origin, such as chicle,crown gum, nispero, rosidinha, jelutong, pendare, perillo, niger gutta,tunu, etc.; masticatory substances of synthetic origin such asbutadiene-styrene polymer, isobutyleneisoprene copolymer, paraffin,petroleum wax, polyethylene, polyisobutylene, polyvinylacetate, etc.

A variety of traditional ingredients may be incorporated in the gumbase, sucb as plasticizers of softeners. Examples of these ingredientsinclude lanolin, stearic acid, sodium stearate, potassium stearate,glyceryl triacetate, glycerine, lecithin, glyceryl monostearate and thelike. Natural waxes, petroleum waxes, polyurethyane waxes, paraffinwaxes and microcrystalline waxes may also be incorporated into the gumbase to obtain a variety of desirable textures and consistencyproperties. Mixtures of these traditional ingredients are alsocontemplated. These traditional ingredients are generally employed inamounts of up to about 30% by weight, and preferably, in amounts of fromabout 3% to about 20% by weight of the final chewing gum product.

Mineral fillers may include aluminum hydroxide, alumina, aluminumsilicate, titanium dioxide, talc, calcium carbonate, tricalciumphosphate, and mixtures thereof.

The flavoring which can be included in the chewing gum compositions madeaccording to this invention can comprise one or more natural and/orsynthetic flavors and/or oils derived from plants, leaves, flowers andfruit. Representative flavors and oils of these types include acids suchas adipic, succinic and fumaric acid; citrus oils cuch as lemon oil,orange oil, lime oil and grapefruit oil; fruit essences, such as appleessence, pear essence, peach essence, strawberry essence, apricotessence, raspberry essence, cherry essence, plum essence and pineappleessence; essential oils cuch as peppermint oil, spearmint oil, bay oil,anise oil, oil of nutmeg, oil of sage, oil of bitter almonds, cassia oiland methylsalicylate (oil of wintergreen). Various synthetic flavors,such as those for a mixed fruit, may also be incorporated in the chewinggum with or without conventional preservatives.

The vehicle or carrier in a tablet or lozenge is a non-cariogenic solidwater soluble polyhydric alcohol (polyol) such as mannitol, xylitol,sorbitol, maltitol, a hydrogenated starch hydrolysate, for exampleLycasin, hydrogenated disaccharides and hydrogenated polysaccharides, inan amount of about 90-98% by weight of the total composition. Solidsalts such as sodium bicarbonate, sodium chloride, potassium bicarbonateor potassium chloride may totally or partially replace the polyolcarrier.

Tableting lubricants, in minor amounts of about 0.1 to 5% by weight, maybe incorporated into the tablet or lozenge formulation to facilitate thepreparation of both the tablets and lozenges. Suitable lubricantsinclude vegetable oils such as coconut oil, magnesium stearate, aluminumstearate, talc, starch and Carbowax

Lozenge formulations may contain about 2% gum as barrier agent toprovide a shiny surface as opposed to a tablet which has a smooth finishSuitable non-cariogenic gums include polycarboxylates such as Kappacarrageenan, carboxymethyl cellulose, hydroxyethyl cellulose, Gantrezand the like.

The lozenge or tablet may optionally be coated with a coating materialsuch as wax, shellac, sodium carboxymethyl cellulose,polyethylene/maleic anhydride copolymer or kappacarrageenan to furtherincrease the time it takes the tablet or lozenge to dissolve in themouth. The uncoated tablet or lozenge is slow dissolving, providing asustained release rate of active ingredients of about 3 to 5 minutes.Accordingly, the solid dose tablet and lozenge composition of thisinvention affords a relatively longer time period of contact with theactive ingredients.

Examples of lozenge formulations which may be employed in the method ofthe present invention are as follows:

    ______________________________________                                                        parts by weight                                               ______________________________________                                        Sorbitol        75-98                                                         Corn Syrup       1-20                                                         Flavor Oil      0.1-1.0                                                       Tablet Lubricant                                                                              0.1-5.0                                                       Oligosaccharide 0.01-5                                                        Water           0.01-0.2                                                      ______________________________________                                    

Various other materials may be incorporated in the oral preparations ofthis invention such as whitening agents, preservatives, for examplephenol, thymol, methyl salicylate, hexylresorcenol, silicones,chlorophyll compounds, anticalculus agents and/or ammoniated materialsuch as urea diammonium phosphate, and mixtures thereof. Theseadjuvants, where present, are incorporated in the preparations inamounts which do not substantially adversely affect the properties andcharacteristics desired.

Any suitable flavoring of sweetening material may also be employed.Examples of suitable flavoring constituents are flavoring oils, e.g. oilof spearmint, peppermint, wintergreen, sassafras, clove, sage,eucalyptus, marjoram, cinnamon, lemon and orange, and methyl salicylate.Suitable sweetening agents include sucrose, lactose, maltose, sorbitol,xylitol, sodium cyclamate, perillartine, APM(aspartyl phenyl alanine,methyl ester), sodium saccharine and the like. Suitably, flavor andsweetening agents may together comprise from about 0.1% to 5% more ofthe preparation.

The following examples are further illustrative of the nature of thepresent invention, but it is understood that the invention is notlimited thereto. All amounts and proportions referred to herein and inthe appended claims are by weight and temperatures are in degrees Cunless otherwise indicated.

Example 1 Mouthrinse

    ______________________________________                                        Mouthrinse                                                                    Ingredient         Weight %                                                   ______________________________________                                        Ethyl Alcohol      10.0                                                       Glycerol           10.0                                                       Flavor             0.4                                                        Sodium Saccharin   0.03                                                       Pluronic F 108     2.0                                                        Cetyl Pyridinium Chloride                                                                        0.05                                                       Lactodifucotetraose                                                                              .1                                                         Water              Q.S. to 100                                                ______________________________________                                    

A mouthrinse composition is prepared according to Example 1. Pluronic F108 is a nonionic surfactant block copolymer of polyoxyethylene andpolyoxypropylene. The mouthrinse is introduced into the oral cavity andmixed therethrough thereby reducing adherence of S. pyogenes toepithelial pharyngeal and oral cells.

Example 2

    ______________________________________                                        Lozenge                                                                       Ingredient       Weight %                                                     ______________________________________                                        Sorbitol         97.2                                                         Corn Syrup       2                                                            Flavor Oil       0.5                                                          Magnesium Stearate                                                                             0.15                                                         Lactodifucotetraose                                                                            0.1                                                          Water q.s        0.05                                                         ______________________________________                                    

A lozenge composition is prepared according to Example 2. The lozenge isintoduced into the oral cavity and dissolved there, thereby reducingadherence of S. pyogenes to epithelial pharyngeal and oral cells.

Example 3 Chewing Gum

    ______________________________________                                        Chewing Gum                                                                   Ingredients      Parts by weight                                              ______________________________________                                        Gum Base         30.0                                                         Sorbitol         42.5                                                         Mannitol         4.0                                                          70% Sorbitol in H.sub.2 O                                                                      16.5                                                         Glycerin         5.0                                                          Lactodifucotetraose                                                                            0.25                                                         Flavoring        q.s. 100                                                     ______________________________________                                    

A chewing gum composition is prepared according to Example 3. Thechewing gum composition is introduced into the oral cavity andmasticated thereby reducing adherence of S. pyogenes to epithelialpharyngeal and oral cells.

Example 4 Chewing Gum

    ______________________________________                                        Chewing Gum                                                                   Ingredients     parts by weight                                               ______________________________________                                        Gum Base        25.00                                                         Lecithin        .5                                                            Softeners       9.6                                                           Mannitol        15.00                                                         Flavor          2.6                                                           Lactodifucotetraose                                                                           0.35                                                          Sorbitol        46.95                                                                         100.00                                                        ______________________________________                                    

A chewing gum composition is prepared according to Example 4. Thechewing gum composition is introduced into the oral cavity andmasticated thereby reducing adherence of S. pyogenes to epithelialpharyngeal and oral cells.

The above chewing gum formulations are exemplary only. Many additionalformulations are described in the prior art, and in carrying out thisinvention, such formulations can be employed.

We claim:
 1. A method of inhibiting adherence of Streptococcus pyrogenesto epithelial pharyngeal and oral cells comprising preparing aformulation containing an oligosaccharide containing at least one fucosemoiety or a galactose moiety which is free of N-acetylneuraminyl lactoseand contacting said cells in the oral cavity with said formulation.
 2. Amethod of inhibiting adherence to epithelial pharyngeal and oral cellsas in claim 1 wherein said oligosaccharide contains at least one fucosemoiety.
 3. A method of inhibiting adherence to epithelial pharyngeal andoral cells as in claim 1 wherein said oligosaccharide contains at leastone galactose moiety free from digalactose and n-acetylneuraminyllactose.
 4. A method of inhibiting adherence to epithelial pharyngealand oral cells as in claim 2 wherein said oligosaccharide is selectedfrom the group consisting of B trisaccharide, A tetrasaccharide,fucosyllactose, and lactodifucotetraose.
 5. A method of inhibitingadherence to epithelial pharyngeal and oral cells as in claim 3 whereinsaid oligosaccharide is lacto-N-tetrose.
 6. A mouthrinse composition forinhibiting adherence of Streptococcus pyogenes to epithelial pharyngealand oral cells comprising 0.1 to 5.0% by weight of an oligosaccharidecontaining at least one fucose moiety or a galactose moiety which isfree from digalactose and n-acetylneuraminyl lactose in a vehiclecomprising a non-toxic alcohol and water.
 7. A mouthrinse composition asin claim 6 wherein said oligosaccharide contains at least one fucosemoiety.
 8. A mouthrinse composition as in claim 6 wherein saidoligosaccharide contains at least one galactose moiety free fromdigalactose and n-acetylneuraminyl lactose.
 9. A mouthrinse compositionaccording to claim 7 wherein said oligosaccharide is selected from thegroup consisting of B trisaccharide, A tetrasaccharide, fucosyllactose,and lactodidifucotetraose.
 10. A mouthrinse composition as in claim 8wherein said oligosaccharide is lacto-N-tetrose.